1. Field of Invention
This invention concerns a method for production of high amounts of carotenoids in plants. In particular, the invention concerns carotenoid-associated proteins CHRC and CHRD isolated from corollas of Cucumis sativus, CHRC deduced sequence, the molecular cloning, cDNA, RNA and a homologous gene involved in expression of CHRC. The Cucumis sativus corollas proteins were found to be involved and to regulate carotenoid accumulation and sequestration in chromoplasts. Invention additionally concerns a new CHRC promoter able to direct expression of foreign genes. The cloned promoter comprises approximately 3.5 kb of the CHRC upstream region.
2. Background Art and Related Art Disclosures
Carotenoids are very important dietary components in animals and are also important for plants as they are essential for plant photosynthesis. In humans, they are essential dietary components, which possess anticancer activity and protect against heart and age related diseases. Commercially, they are used-to color food products. In flowers and plants they are responsible for their pigmentation. In photosynthetically active organisms, carotenoids are essential to the proper functioning of the light-harvesting apparatus and they determine color of fruits and flowers. Moreover, carotenoid synthetic pathway leads to the synthesis of key growth regulators.
In plants, carotenoids are accumulated in high amounts chromoplasts, that are carotenoid-containing plastids responsible for the yellow, orange and red colors of petals, fruits and some roots in various plant species. Information on the structural organization of chromoplasts comes mainly from studies of ripening processes in fruits [Plant Sci. 94:1-17 (1983)]. The disappearance of chlorophyll and accumulation of carotenoids have been shown to parallel fruit maturation. Following the chloroplast to chromoplast conversion thylakoid membranes disintegrate, most of the components of the photosynthetic machine disappear, and a new set of proteins accumulates instead. In pepper fruits containing fibrillar chromoplasts, two very abundant proteins with approximate molecular masses of 35 kDa (ChrB) and 58 kDa (ChrA) have been identified as chromoplast-specific [Plant Mol. Biol. 21:549-554 (1993)]. When their pattern of expression was studied, the former was found to accumulate early and remain throughout ripening, whereas the latter appeared only at the final stage of fruit-color development and was found to be a carotenoid-associated protein [Plant Physiology 91:455-458 (1989)].
Identification of the protein fibrillin in bell peppers as an essential structure component of chromoplast-specific, carotenoid-accumulating lipoprotein structures, termed fibrils, and the isolation of its cDNA and genomic clones were very important steps towards understanding the internal structure of chromoplasts [Plant Cell 6:119-133 (1994)]. Analysis of this gene""s expression in bell pepper revealed that both the protein and the transcript accumulate in parallel to fruit ripening. The bell pepper clone, however, did not reveal homologous transcripts in other tissues or plants, ibid.
To date, a number of genes from the carotenoid biosynthetic pathway in fruits have been cloned [Plant Mol. Biol. 27:425-428 (1995); Plant Mol. Biol. 27:1153-1162 (1995)]. Of these, however, only one, encoding capsanthin-capsorubin synthase [Plant Cell 7:1027-1038 (1995); Plant J. 6:45-54 (1994); Curr. Genet. 26:524-527 (1994)], shows fruit-chromroplast-specific expression.
Flower pigmentation due to carotenoid accumulation has also been studied. In Tropaeolum majus corollas, a 30 kDa protein accumulated in parallel to flower development and appeared to be a major and obligatory component of chromoplast fibrils [Protoplasma 157:128-135 (1990)]. Proteins of 30 and 68 kDa were characterized as the main proteins of Viola tricolor chromoplast globules [Plant Cell Res. 1:111-114 (1982)]. In nasturtium flowers, an immunocomplex band of 32 kDa was revealed with fibrillin antibodies [Plant Cell 6:119-133 (1994)]. With respect to genes of the carotenoid biosynthetic pathway, expression of phytoene synthase (PSY) and phytoene desaturase (PDS) in tomato flowers was shown to peak just before anthesis [Plant Cell 5:379-384 (1993)]. The petals and anthers of mature flowers accumulated the highest levels of these transcripts, as compared with other organs. Nevertheless, expression of both PSY and PDS was found not to be flower-specific.
Young green flower buds of Cucumis sativus contain chloroplasts, which are converted to fibrillar chromoplasts as the flower matures. Only chromoplasts are found in the mature yellow corollas [Physiol. Plant 104:321-326 (1994)]. An isolated 35 kDa chromoplast-specific protein (CHRC) from cucumber corollas was shown to be associated with carotenoids. While the above-findings are interesting from the botanical point of view, they do not have a practical utility.
Since carotenoids are so important for both plants and animals it would be of a great advantage to utilize in some way the above findings to achieve and control a higher production or accumulation of carotenoids in plants or cells, or their easy production by bacterial cells.
It is, therefore a primary object of this invention to provide a method for genetic control of production and/or accumulation of high levels of carotenoids in cells of plants, bacteria or other organisms.
One aspect of the current invention is a method for production, accumulation and sequestration of high amounts of carotenoids in plant, bacteria or other cells by molecular and other type manipulations of carotenoid-associated proteins.
Another aspect of the current invention is a gene of which expression produces a protein which controls and is involved in a production, accumulation and sequestration of carotenoids in plant, bacteria or other cells.
Another aspect of the current invention is a method for molecular cloning of chrc gene encoding for the carotenoid-associated protein CHRC from Cucumis sativus. 
Still another aspect of the current invention is a gene containing domain which is homologous to domains present in a variety of chromoplasts containing plants.
Still another aspect of the current invention is a nucleotide sequence (SEQ ID NO:1) of the chrc gene encoding the Cucumis sativus protein CHRC.
Still another aspect of the current invention is an amino acid sequence (SEQ ID NO:2) comprising 322 amino acid of the CHRC protein of Cucumis sativus. 
Still yet another aspect of the current invention is a partly sequenced CHRC promoter having a nucleotide sequence (SEQ ID NO:10).
Another aspect of the current invention is a method for high production of carotenoids using bacterial cells having introduced CHRC gene intracellularly together with genes encoding enzymes for carotenoid biosynthesis.
As used herein:
xe2x80x9cCHRCxe2x80x9d means chromoplast-specific carotenoid-associated protein.
xe2x80x9cCHRDxe2x80x9d means a minor chromoplast-specific protein of about 14 kD isolated from cucumber corolla chromoplasts.
xe2x80x9cAnthesisxe2x80x9d means the flowering period in flowers and plants.
xe2x80x9cCorollaxe2x80x9d means collectively the petals and flowers.
xe2x80x9cThylakoidxe2x80x9d means a membranous lamella of protein and lipid implant chloroplasts where the photochemical reactions of photosynthesis take place.
xe2x80x9cGibberellinsxe2x80x9d or xe2x80x9cGAxe2x80x9d means plant hormones that regulate various aspects of plant growth and development, such as germination, cell growth, stem elongation, flower and fruit development and pigmentation.
xe2x80x9cGibberellin A3xe2x80x9d or xe2x80x9cGA3xe2x80x9d means a hormone which up-regulates production of CHRC protein expression.